GSM7179346: BS_ddm1-FLA_rep2; Arabidopsis thaliana; Bisulfite-Seq
Whole-genome bisulfite-seq: Nuclear DNA was extracted from 12 days old seedlings as described in DRIP/DRIP-seq section and sonicated to 250bp with COVARIS. After that Bisulfite conversion and DNA recovery was performed with EZ DNA Methylation-Gold Kit (ZYMO Research) by following the manufacturer instructions. Finally, the sequencing library is made using the EpiArt DNA Methylation Library Kit for Illumina V3 (Vazyme, NE103) by following the manufacturer instructions. ChIP-seq: To gain the nucleosome resolution, native H3K9me2 ChIP without crosslink and sonication was performed as described in (Bernatavichute et al., 2008), with some modifications. In brief, nuclei were extracted from 12 days old seedlings with Honda buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM HEPES–KOH [pH 7.4], 10 mM MgCl2, 0.5% Triton X-100, 5 mM DTT, and protease inhibitors [Roche]). Nuclei were suspended in 1X MNAse buffer (50mM TriHCl pH 7.6, 5mM CaCl2, 0.1mM PMSF) containing 1X proteinase inhibitor cocktail (Roche). After RNase A (Thermo Scientific™) treatment at 37C for 30 minutes, chromatin was digested with MNase enzyme (NEB) for 6 minutes and the reaction was stopped with EDTA (10mM). Then, the nucleosomes were released with the addition of 0.005% SDS while rotating at 4C for 2-3 hours. After that the supernatant was separated and diluted with dilution buffer (0.1% triton, 50mM NaCl, 0.1mM PMSF, 17mM trisHCl, 3.33mM EDTA, 1x Cocktail inhibitor). Chromatin was pre-cleaned with Protein G beads, and immnoprecipitated with anti-H3K9me2 (Abcam, ab1220) antibodies at 4C overnight. The complexes were incubated with protein G beads (Millipore) at 4C for 3 hours with rotation, and washed three times with ChIP wash buffer having different concentration of NaCl in each wash (50mM TrisHCl pH 7.6, 10mM EDTA, 50/100/150 mM NaCl, 0.1mM PMSF, and 1x Cocktail inhibitor). Then DNA was eluted with elution buffer (0.1%SDS and 0.1M NaHCO3) at 65C for 15 minutes. Proteinase K treatment was performed at 55 C for 2 hour. DNA was recovered after phenol:chloroform extraction. Finally, the sequencing library is made using the TruePrep® DNA Library Prep Kit V2 for Illumina (Vazyme, TD502) by following the manufacturer instructions.